Identification of novel modulators of a schistosome transient receptor potential channel targeted by praziquantel.

Given the worldwide burden of neglected tropical diseases, there is ongoing need to develop novel anthelmintic agents to strengthen the pipeline of drugs to combat these burdensome infections. Many diseases caused by parasitic flatworms are treated using the anthelmintic drug praziquantel (PZQ), employed for decades as the key clinical agent to treat schistosomiasis. PZQ activates a flatworm transient receptor potential (TRP) channel within the melastatin family (TRPMPZQ) to mediate sustained Ca2+ influx and worm paralysis. As a druggable target present in many parasitic flatworms, TRPMPZQ is a promising target for a target-based screening campaign with the goal of discovering novel regulators of this channel complex. Here, we have optimized methods to miniaturize a Ca2+-based reporter assay for Schistosoma mansoni TRPMPZQ (Sm.TRPMPZQ) activity enabling a high throughput screening (HTS) approach. This methodology will enable further HTS efforts against Sm.TRPMPZQ as well as other flatworm ion channels. A pilot screen of ~16,000 compounds yielded a novel activator of Sm.TRPMPZQ, and numerous potential blockers. The new activator of Sm.TRPMPZQ represented a distinct chemotype to PZQ, but is a known chemical entity previously identified by phenotypic screening. The fact that a compound prioritized from a phenotypic screening campaign is revealed to act, like PZQ, as an Sm.TRPMPZQ agonist underscores the validity of TRPMPZQ as a druggable target for antischistosomal ligands.

In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major.

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

Immunomodulatory action of excretory-secretory products of Angiostrongylus cantonensis in a mouse tumour model.

Excretory-secretory products (ESPs) of parasitic helminths are well known to exert immunostimulation and immunomodulation in hosts. Immune regulation plays a key role in anti-tumour therapy. The present study explored the anti-tumour effect of ESPs released by Angiostrongylus cantonensis. In Hepa1-6 mouse tumour models, ESPs significantly reduced tumour growth. Tumour-bearing mice treated with ESPs had significantly higher CD3+, CD4+, and CD8+ T cell counts than those treated with Freund's adjuvant. In vitro, human hepatocarcinoma HepG2 cells, human lung cancer A549 cells, and normal human liver HL-7702 cells were co-incubated with ESPs for 24 h and 48 h. ESPs significantly accelerated HepG2 apoptosis but had no inhibitory effect on the proliferation of A549 and HL-7702 cells. Apoptotic HepG2 cells displayed condensed nuclei, apoptotic bodies, and swollen endoplasmic reticulum (ER). Expression of the endoplasmic reticulum stress (ERS)-related factors activating transcription factor 6 (ATF6) and C/EBP-homologous protein (CHOP) in HepG2 cells increased with increasing ESP concentration and treatment time. Calreticulin (CRT) is a key effector protein of ESPs, and recombinant calreticulin (rCRT) was produced in BL21 Escherichia coli (E. coli). In contrast to ESPs, rCRT markedly reduced the proliferation of HepG2 cells. The expression levels of ATF6 and CHOP in HepG2 cells treated with 30 µg/mL rCRT significantly increased at 48 h. Notably, these findings synergistically suggest that ESPs and rCRT are promising candidates for anti-tumour immunotherapy.

Deep phosphoproteome analysis of Schistosoma mansoni leads development of a kinomic array that highlights sex-biased differences in adult worm protein phosphorylation.

Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a 'footprint' of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.

Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii.

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism.

Adjuvant-free immunization with infective filarial larvae as lymphatic homing antigen carriers.

Controlled infection with intestinal nematodes has therapeutic potential for preventing the symptoms of allergic and autoimmune diseases. Here, we engineered larvae of the filarial nematode Litomosoides sigmodontis as a vaccine strategy to induce adaptive immunity against a foreign, crosslinked protein, chicken egg ovalbumin (OVA), in the absence of an external adjuvant. The acylation of filarial proteins with fluorescent probes or biotin was not immediately detrimental to larval movement and survival, which died 3 to 5 days later. At least some of the labeled and skin-inoculated filariae migrated through lymphatic vessels to draining lymph nodes. The immunization potential of OVA-biotin-filariae was compared to that of an OVA-bound nanoparticulate carrier co-delivered with a CpG adjuvant in a typical vaccination scheme. Production of IFNγ and TNFα by restimulated CD4+ cells but not CD8+ confirmed the specific ability of filariae to stimulate CD4+ T cells. This alternative method of immunization exploits the intrinsic adjuvancy of the attenuated nematode carrier and has the potential to shift the vaccination immune response towards cellular immunity.

Transcriptome analysis of Globodera pallida from the susceptible host Solanum tuberosum or the resistant plant Solanum sisymbriifolium.

A transcriptome analysis of G. pallida juveniles collected from S. tuberosum or S. sisymbriifolium 24 h post infestation was performed to provide insights into the parasitic process of this nematode. A total of 41 G. pallida genes were found to be significantly differentially expressed when parasitizing the two plant species. Among this set, 12 were overexpressed when G. pallida was parasitizing S. tuberosum and 29 were overexpressed when parasitizing S. sisymbriifolium. Out of the 12 genes, three code for secretory proteins; one is homologous to effector gene Rbp-4, the second is an uncharacterized protein with a signal peptide sequence, and the third is an ortholog of a Globodera rostochiensis effector belonging to the 1106 effector family. Other overexpressed genes from G. pallida when parasitizing S. tuberosum were either unknown, associated with a stress or defense response, or associated with sex differentiation. Effector genes namely Eng-1, Cathepsin S-like cysteine protease, cellulase, and two unknown genes with secretory characteristics were over expressed when G. pallida was parasitizing S. sisymbriifolium relative to expression from S. tuberosum. Our findings provide insight into gene regulation of G. pallida while infecting either the trap crop S. sisymbriifolium or the susceptible host, S. tuberosum.

Re-targeting of a plant defense protease by a cyst nematode effector.

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector-coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two-hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain-like cysteine protease (PLCP) 'Responsive to Dehydration 21A' (RD21A), which has been shown to function in the plant defense response. Activity-based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re-localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two-hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector-mediated re-localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense-inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.

Antifilarial activity of azadirachtin fuelled through reactive oxygen species induced apoptosis: a thorough molecular study on Setaria cervi.

Efficacious therapeutic strategies against lymphatic filariasis are always sought after. However, natural products are a promising resource for developing effective antifilarial agents. Azadirachtin, a significant tetranortriterpenoid phytocompound found in Azadirachta indica, was evaluated in vitro for antifilarial potential against the filarial parasite Setaria cervi. Dye exclusion and MTT assay confirmed the antifilarial potential of azadirachtin against S. cervi with a median lethal dose (LC50) of 6.28 µg/ml for microfilariae (mf), and 9.55 µg/ml for adult parasites. Morphological aberrations were prominent in the histological sections of the azadirachtin-exposed parasites. Moreover, alterations in the reactive oxygen species (ROS) parameters in treated parasites were evident. Induction of apoptosis in treated parasites was confirmed by DNA laddering, acridine orange (AO)/ethidium bromide (EtBr) double staining and in situ DNA fragmentation. The downregulation of anti-apoptotic CED-9 and upregulation of proapoptotic EGL-1, CED-4 and CED-3 at both the transcription and translation levels confirmed apoptosis execution at the molecular level. Changes in the gene expressions of nuc-1, cps-6 and crn-1 further clarified the molecular cause of DNA degradation. Furthermore, azadirachtin was found to be non-toxic in both in vitro and in vivo toxicity analyses. Therefore, the experimental evidence detailed the pharmacological effectiveness of azadirachtin as a possible therapeutic agent against filariasis.

Proteogenomic Analyses Revealed Favorable Metabolism Pattern Alterations in Rotifer Brachionus plicatilis Fed with Selenium-rich Chlorella.

Organoselenium have garnered attention because of their potential to be used as ingredients in new anti-aging and antioxidation medicines and food. Rotifers are frequently used as a model organism for aging research. In this study, we used Se-enriched Chlorella (Se- Chlorella), a novel organoselenium compound, to feed Brachionus plicatilis to establish a rotifer model with a prolonged lifespan. The results showed that the antioxidative effect in Se-enriched rotifer was associated with an increase in guaiacol peroxidase (GPX) and catalase (CAT). The authors then performed the first proteogenomic analysis of rotifers to understand their possible metabolic mechanisms. With the de novo assembly of RNA-Seq reads as the reference, we mapped the proteomic output generated by iTRAQ-based mass spectrometry. We found that the differentially expressed proteins were primarily involved in antireactive oxygen species (ROS) and antilipid peroxidation (LPO), selenocompound metabolism, glycolysis, and amino acid metabolisms. Furthermore, the ROS level of rotifers was diminished after Se- Chlorella feeding, indicating that Se- Chlorella could help rotifers to enhance their amino acid metabolism and shift the energy generating metabolism from tricarboxylic acid cycle to glycolysis, which leads to reduced ROS production. This is the first report to demonstrate the anti-aging effect of Se- Chlorella on rotifers and to provide a possible mechanism for this activity. Thus, Se- Chlorella is a promising novel organoselenium compound with the potential to prolong human lifespans.

Identification and function of FAR protein family genes from a transcriptome analysis of Aphelenchoides besseyi.

Motivation: The rice white tip nematode (RWTN) Aphelenchoides besseyi is a migratory plant parasitic nematode that infects the aboveground parts of plants. Fatty acid- and retinoid-binding (FAR) proteins are nematode-specific proteins that are involved in many important biological processes. Genes encoding FAR proteins have been identified in many species of nematodes, which indicated that nematodes may produce more than one type of FAR protein. The main goal of this study is to find new molecular targets including new far genes that will help control RWTN, and reduce the economic damage caused by RWTN. Results: Two RWTN populations with different levels of pathogenicity and reproduction were sequenced and analyzed with next-generation sequencing. 17 087 transcripts were annotated using six databases and 1696 differentially expressed genes (DEGs) were identified between the two RWTN populations. Seven new Ab-far genes were identified from the transcriptome data of the two RWTN populations which is the first to identify multiple far genes in plant parasitic nematodes. This study is the first to identify far genes in the nervous system of nematodes and the first to report a transcriptome sequencing analysis of different RWTN populations. The results help elucidate the genes related to parasitism and pathogenicity and also contribute to the identification of new target genes and development of new methods to control RWTN. Availability and implementation: Our data are publicly available at Sequence Read Archive (SRA) database and GenBank database. Supplementary information: Supplementary data are available at Bioinformatics online.

Evaluating the longevity of surgically extracted Xenopus laevis oocytes for the study of nematode ligand-gated ion channels.

Xenopus laevis oocytes have been extensively used as a heterologous expression system for the study of ion channels. While used successfully worldwide as tool for expressing and characterizing ion channels from a wide range of species, the limited longevity of oocytes once removed from the animal can pose significant challenges. In this study, we evaluate a simple and useful method that extends the longevity of Xenopus oocytes after removal from the animal and quantitatively assessed the reliability of the electrophysiological date obtained. The receptor used for this study was the UNC-49 receptor originally isolated from the sheep parasite, Haemonchus contortus. Overall, we found that immediate storage of the ovary in supplemented ND96 storage buffer at 4 °C could extend their use for up to 17 days with almost 80% providing reliable electrophysiological data. This means that a single extraction can provide at least 3 weeks of experiments. In addition, we examined 24-day-old oocytes (week 4) extracted from a single frog and also obtained reliable data using the same approach. However, 50% of these oocytes were usable for full dose-response experiments. Overall, we did find that this method has the potential to significantly extend the use of single oocyte extractions for two-electrode voltage clamp electrophysiology.

Biological significance of phosphoenolpyruvate carboxykinase in a cestode parasite, Raillietina echinobothrida and effect of phytoestrogens on the enzyme from the parasite and its host, Gallus domesticus.

Phosphoenolpyruvate carboxykinase (PEPCK) is involved in glycolysis in the cestode parasite, Raillietina echinobothrida; whereas, it executes a gluconeogenic role in its host, Gallus domesticus. Because of its differing primary function in the cestode parasite and its host, this enzyme is regarded as a plausible anthelmintic target. Hence, the biological significance of PEPCK in the parasite was analysed using siRNA against PEPCK from R. echinobothrida (RePEPCK). In order to find out the functional differences between RePEPCK and GdPEPCK (PEPCK from its host, G. domesticus), PEPCK genes from both sources were cloned, over-expressed, characterized, and some properties of the purified enzymes were compared. RePEPCK and GdPEPCK showed a standard Michaelis-Menten kinetics with K mapp of 46.9 and 22.9 µ m, respectively, for phosphoenolpyruvate and K mapp of 15.4 µ m for oxaloacetate in GdPEPCK decarboxylation reaction. Here, we report antagonist behaviours of recombinant PEPCKs derived from the parasite and its host. In search of possible modulators for PEPCK, few phytoestrogens were examined on the purified enzymes and their inhibitory constants were determined and discussed. This study stresses the potential of these findings to validate PEPCK as the anthelmintic drug target for parasitism management.

Identification of AcAP5 as a novel factor Xa inhibitor with both direct and allosteric inhibition.

Ancylostoma caninum anticoagulant peptide 5 (AcAP5) is a potent inhibitor for coagulation factor Xa (FXa). Previous studies show that AcAP5 binds to FXa at the active site, and/or the exosite. The active site-binding contributes to direct blocking of FXa catalytic activity, but the effect of exosite-binding and the underlying mechanism remain unknown. To investigate whether and how the exosite-binding affects FXa function, we prepared several AcAP5 mutants with modifications to the active site-binding or exosite-binding region. Their FXa-inhibiting and anticoagulant activities were examined both in vitro and in rabbit plasma, and the interactions with FXa were analyzed using in silico molecular modeling, docking, and molecular dynamics simulation. Mutants abolishing either active site- or exosite-binding resulted in a dramatic decrease in their anti-FXa and anticoagulant activities. Elongation of AcAP5 exosite-binding region also impaired the FXa-inhibiting activity. Computational analysis demonstrated that the conformation of FXa becomes more rigid due to exosite-binding with AcAP5, which consequently affects its catalytic activity. Our results suggest that both active site- and exosite-binding contribute to the FXa inhibitory activity of AcAP5.

Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica.

Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses. However, the existence of PLSCRs and their biological functions in planarian are still unknown at present. In this study, a new member of PLSCRs was identified in planarian Dugesia japonica (D. japonica), named DjPLSCR. The sequence analysis revealed that it contains an opening reading frame consisting of 726bp encoding a putative protein of 241 amino acids with a predicted molecular mass of ~28.7kDa and an isoelectric point of 6.21. Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and ß-glucan], suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians.

Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae.

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.

Analysis of the Transcriptome of the Infective Stage of the Beet Cyst Nematode, H. schachtii.

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii.

Molecular cloning and characterization of a phospholipid hydroperoxide glutathione peroxidase gene from a blood fluke Schistosoma japonicum.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a major antioxidant enzyme and plays critical roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. A full-length cDNA sequence corresponding to GPx gene from Schistosoma japonicum (designated SjGPx) was isolated and characterized. SjGPx contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3'-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Phylogenetic analysis revealed that the SjGPx was highly related to the other PHGPx-related members, and clustered into the trematode subclade II. Semi-quantitative reverse transcription PCR and western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. We observed a 53.86% reduction in total GPx activity and the eggs severely deformed. Oxidative stimulation of viable worms with H2O2 or paraquat resulted in 1.6- to 2.1-fold induction of the GPx activity. Our results revealed that the SjGPx protein is selenium-dependent PHGPx, which might actively participate in the detoxification of oxidative damage during egg production.

Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.